Regulation of protein synthesis at the level of mRNA translation is an important mechanism involved in: 1) viral shut-off of host protein synthesis, 2) inhibition of viral replication by interferon, 3) regulation of correct developmental sequences, and 4) coordination of heme-globin biosynthesis during red blood cell maturation. Since translational regulation of protein synthesis can only be observed in unfractionated systems, we have extended our studies with highly purified translational components to the normal sequence of protein synthesis initiation and its regulation by hemin in reticulocyte lysate. Using radiolabeled initiation factors, highly specific inhibitors and quantitative techniques for the identification of mRNA and Met-tRNAf, we have identified, determined the composition of and order of formation of specific preinitiation complexes formed during the initiation process. We have, therefore, been able to apply crossover analysis to the mechanism of hemin regulated translational control. Evidence to date suggests that activation of a highly specific cAMP-independent protein kinase for the initiator Met-tRNAf binding protein eIF-2 inhibits ribosomal binding of Met-tRNAf by interference with eIF-2 recycling and altered preinitiation complex stability.